Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex)

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites.

The studies of m 2 G10 in tRNA and Trm11 described above have been reported over the past two decades. However, one important question remains: why is the m 2 G modification only observed in a limited number of tRNA species in S. cerevisiae (Figure 2 and References [42,43]). In this paper, the numbering of position in tRNA is according to Reference [44]. As shown in Figure 2, the recognition site(s) of S. cerevisiae Trm11-Trm112 in tRNA cannot simply be predicted from the sequences of modified and unmodified tRNAs. Fortunately, during the course of this study, a structural model of the Trm11-Trm112 complex was proposed [9]. In the current study, we performed biochemical studies to clarify the required elements in tRNA for methylation by the Trm11-Trm112 complex and discuss the experimental results based on the proposed structural model. Comparison of S. cerevisiae tRNA sequences. The sequences of D-arms, anticodonloops, variable regions and T-loops in S. cerevisiae tRNAs are compared. The abbreviations are as follows; m 1 G, 1-methylguanosine; D, dihydrouridine; I, inosine; m 1 I, 1-methylinosine; T, thymidine; Ψ, pseudouridine; i 6 A, N 6 -isopetenyladenosine; m 7 G, 7-methylguanosine; m 1 A, 1methyladenosine; U*, U-modification; X, unidentified modification; Gm, 2 -O-methylguanosine; Um, 2 -O-methyluridine; ac 4 C, 4-acetylcytidine; m 5 C, 5-methylcytidine; m 3 C, 3-methylcytidine; t 6 A, N 6 -threonylcarbamoyladenosine; and yW, wyosine. The position of a modification caused by Trm11-Trm112 (m 2 G10) is highlighted in red. The sequences in the aminoacyl-stem, anticodon-stem and T-stem are not shown.

Co-Expression of Trm11 and Trm112 in Escherichia coli Cells and Purification
In our previous study, we reported the production of a Trm11-Trm112 complex in a wheat germ cell-free expression system to investigate whether the cell-free system can synthesize a multiple protein complex [45]. In the current study, we constructed a coexpression system for Trm11-Trm112 in Escherichia coli cells and purified Trm11-Trm112 (Supplementary Figure S1). The details are described in Supplementary File S1.

Measurement of Methyltransferase Activity of Trm11-Trm112 Complex
We measured the methyltransferase activity of purified Trm11-Trm112 in several conditions and noticed that the diluted enzyme rapidly decreased in activity (Supplementary Figure S2). During the course of this study, a time course assay of Trm11-Trm112 activity toward a tRNA Ile AAU transcript was reported and the methyl-transfer activity of this Trm11-Trm112 was also observed to decrease rapidly [9]. Thus, this phenomenon is not caused by tRNA species but is probably caused by the dissociation of Trm11-Trm112 subunits in the reaction mixture. When 0.10 µM Trm11-Trm112, 10.0 µM yeast tRNA Phe transcript and 20.1 µM AdoMet (20 µM non-radioisotope labeled AdoMet and 0.06 µM 3 H-AdoMet) were incubated at 30 • C, 3 H-methyl group incorporation into the tRNA Phe transcript showed linearity for the initial 2 min (Supplementary Figure S2). Therefore, in this study, we measured the initial velocities of the methyl-transfer reaction for 2 min periods in the above conditions. Furthermore, it should be mentioned that Trm11-Trm112 strictly recognizes the CCA terminal region of tRNA, as described later in this paper. Therefore, we prepared tRNA transcript using 2 -O-methylated DNA for the construction of a template to avoid adding extra nucleotide(s) to the CCA terminal region. To visualize the methyl group incorporation, 14 C-AdoMet was used. Details are described in the Materials and Methods section.

Precursor tRNA Phe Is Not Methylated by the Trm11-Trm112 Complex
As described in the Introduction, the substrate tRNA recognition mechanism of Trm11-Trm112 cannot simply be predicted by the sequences of tRNAs from S. cerevisiae. In S. cerevisiae, an intron in precursor tRNA is removed in the cytoplasm [46,47] and precursor tRNA is repeatedly transported between cytoplasm and nucleus [46,48,49]. It should be mentioned that Trm11 is localized in the cytoplasm [4]. Furthermore, although the 5 -cap structure is attached to four precursor tRNAs (tRNA Ile UAU , tRNA Leu CAA , tRNA Lys UUU and tRNA Trp CCA ) during the transport process, the attachment of the cap structure to precursor tRNA Phe has not been reported [49]. At the beginning of this study, we assumed that intron, and/or leader and trailer sequences of precursor tRNAs might have an effect on the substrate tRNA recognition by Trm11-Trm112. To address this issue, we prepared four types of tRNA Phe transcripts ( Figure 3A). Transcript 1 is the mature-size tRNA Phe transcript. Transcript 2 has an intron at the canonical position between positions 38 and 39. Transcript 3 has 5 -leader and 3 -trailer sequences. Transcript 4 has an intron, and 5 -leader and 3 -trailer sequences. These transcripts were incubated at 30 • C for 2 min with Trm11-Trm112 and 14 C-AdoMet, and then, RNAs were recovered from phenol-chloroform treatment. The RNA samples were loaded onto a 10% polyacrylamide gel containing 7 M urea (PAGE (7 M urea)), and an autoradiogram of the gel obtained ( Figure 3B).
As shown in Figure 3B, the 14 C-methyl group was incorporated only into the maturesize tRNA Phe transcript. Thus, precursor tRNAs are not substrates for Trm11-Trm112. The results suggest that the m 2 G10 modification by Trm11-Trm112 is one of the final events in tRNA maturation in S. cerevisiae cells. Furthermore, because the presence of an intron affects methylation by Trm11-Trm112, one of the recognition sites of Trm11-Trm112 is predicted to be the anticodon-loop. Moreover, because the 5 -leader and 3 -trailer sequences negatively affects methylation, Trm11-Trm112 is predicted to recognize the end(s) of the aminoacyl-stem in addition to the anticodon-loop of tRNA.

Deletion and Stem-Disruptant Mutants of tRNA Phe Transcripts Are Not Methylated by Trm11-Trm112
To clarify the recognition site(s) of Trm11-Trm112, we prepared twelve mutant tRNA Phe transcripts ( Figure 4A). Nine transcripts (transcripts 5-13) were deletion mutants and three transcripts (transcripts [14][15][16] were disruptant mutants of the stems. To our surprise, none of mutants were methylated by Trm11-Trm112 ( Figure 4B,C). The experiments using precursor tRNA transcripts ( Figure 3) suggest that the anticodon-loop and aminoacyl-stem are the recognition sites of the Trm11-Trm112 complex. However, with transcripts 6, 8, 9, 11, 14 and 16, their anticodon-arm and aminoacyl-stem are not mutated. These results suggest that the recognition sites of the Trm11-Trm112 complex are plentiful and are dispersed through the L-shaped tRNA structure.

The CCA Terminus Is Essential for Methylation by Trm11-Trm112
During the course of this study, we solved the crystal structure of T. kodakarensis Trm11 [8], which catalyzes the formation of m 2 G10 and m 2 2 G10 in tRNA. Thermococcus kodakarensis Trm11 does not require a partner subunit (Trm112) for activity [8]. In the study, we reported that the THUMP domain in T. kodakarensis Trm11 recognizes the CCA terminus in tRNA [8]. The recognition of the 3 -end of substrate tRNA by a THUMP domain has also been reported for other tRNA modification enzymes [8][9][10]19,20]. For example, the crystal structure of complex of Thermotoga maritima ThiI (tRNA 4-thiouridine synthetase) and truncated tRNA revealed that the THUMP domain in ThiI captures the 3 -terminal region in tRNA [20]. Because eukaryotic Trm11 also possesses a THUMP domain [4], we considered that the 3 -terminal region of tRNA may be one of the recognition sites of Trm11-Trm112. To examine this idea, we prepared ten mutant tRNA Phe transcripts, in which the CCA sequence was mutated or deleted ( Figure 5).
When A76 was replaced by G, U or C (transcripts 17, 18 or 19), the methyl group acceptance activity was considerably decreased. These results reveal that the adenine base at position 76 is important for methylation by Trm11-Trm112. In contrast, cytosine bases at positions 74 and 75 are not important because mutant transcripts 20 and 21 were well methylated by Trm11-Trm112. However, the presence of nucleotides at positions 74 and 75 themselves is required for efficient methylation by Trm11-Trm112 because the methyl group acceptance activities of deletion mutants (transcripts 24, 25 and 26) were considerably decreased. These results show the importance of A76 and the ribose-phosphate backbone of the CCA terminus for recognition by Trm11-Trm112. During the course of this study, the methyl-transfer activity of Trm11-Trm112 towards a CCA deletion mutant of tRNA Ile transcript was reported [9]. These results also highlight the importance of the CCA terminus for methylation by Trm11-Trm112 [9]. The deletion mutant tRNA Phe transcripts were incubated with Trm11-Trm112 and 14 C-AdoMet and then loaded onto a 10% polyacrylamide gel containing 7 M urea. After the electrophoresis, the gel was stained with methylene blue and dried (left). An autoradiogram of the gel was obtained (right). One full-length tRNA Phe transcript was methylated by Trm11-Trm112. (C) Methylation of the stem-disruptant mutant tRNA Phe transcripts was analyzed using the same method described in panel (B). Disruption of D-stem, anticodon-stem or T-stem abolishes methylation by Trm11-Trm112.

Requirement of the G10-C25 Base Pair and Interaction between the D-Arm and T-Arm
In the L-shaped tRNA Phe structure, several tertiary base pairs are formed (blue dotted lines in Figure 6A and Reference [36]). We considered whether tertiary base pairs might be involved in the efficiency of methylation by Trm11-Trm112 because several tertiary base pairs are formed around the methylation site G10. We prepared eleven tRNA Phe transcript variants and measured their methyl group acceptance activities ( Figure 6B). Contrary to our expectation, the tertiary base pairs around G10 are not essential. However, when U55 and C56 were substituted by A55 and A56, the methyl-group acceptance activity was lost (transcript 33). This result shows that the interaction between the D-arm and T-arm is essential for methylation by Trm11-Trm112. Furthermore, when C25 was substituted by A or U (transcripts 34 and 35), the methyl group acceptance activity was nearly completely lost. Thus, these results reveal that the G10-C25 base pair is essential for the methylation by Trm11-Trm112. These results explain a part of the G10 methylation patterns presented in Figure 2. All tRNAs that possess U25 (tRNA Asp , two tRNA Gly and two tRNA Pro ) are not methylated by Trm11-Trm112 in vivo. Thus, U25 acts as a negative element for methylation by Trm11-Trm112. Furthermore, when the G10-G45 tertiary base pair was disrupted, the methyl-group acceptance activity was clearly increased (transcript 36). This result suggests that the G10-G45 tertiary base pair is disrupted during the methyl-transfer reaction mediated by Trm11-Trm112.

Tranfer RNAs with Short Variable Region Are Not Methylated by Trm11-Trm112 Complex
In the L-shaped tRNA, the variable region makes contact with the D-arm, which contains the methylation site G10 ( Figure 6A). To address the effect of the variable region on methylation by Trm11-Trm112, we prepared five tRNA Phe variants (transcripts 38-42 in Figure 7). When A44 was substituted by U, the methyl group acceptance activity was slightly increased. This result shows that the formation of the G26-U44 base pair does not inhibit methylation by Trm11-Trm112. In contrast, when the length of the variable region was shortened, the methyl-group acceptance activity was completely lost (transcripts 39 and 40). This result explains a part of the m 2 G10 modification patterns observed in S. cerevisiae tRNAs ( Figure 2). Six tRNA species (tRNA Asp , tRNA Glu , two tRNA Gly and two tRNA His ) in S. cerevisiae tRNAs possess a short variable region, which is composed of three or four nucleotides, and these tRNAs do not possess the m 2 G10 modification. Thus, the presence of a short variable region in tRNA acts as a negative element for methylation by Trm11-Trm112. Furthermore, we investigated whether the sequence of the variable region might have an effect on methylation by Trm11-Trm112. The variable region of tRNA Phe was replaced by that of tRNA Pro or tRNA Val AAC1 (transcripts 41 and 42 in Figure 7): tRNA Pro and tRNA Val AAC1 are reported to possess unmodified G10 ( Figure 2). Contrary to our expectation, these tRNA transcript variants were methylated by Trm11-Trm112. Thus, Trm11-Trm112 does not distinguish between these sequences of the variable region.

Anticodon-Loop Sequence Is Important for Methylation by Trm11-Trm112
Transfer RNAs, which possess U25 and/or a short variable region, are not methylated by Trm11-Trm112. However, the m 2 G modification patterns of the three tRNA molecular species (tRNA Ala , tRNA Cys and tRNA Val AAC1 ) shown in Figure 2 are not explainable by these properties because these tRNAs possess C25 and a regular size variable region, which is composed of five nucleotides. To verify whether these tRNA transcripts are substrates for Trm11-Trm112, we prepared the tRNA Asn , tRNA Thr , tRNA Trp , tRNA Val AAC1 , tRNA Ala , tRNA Cys , tRNA Pro and tRNA Val UAC transcripts and tested their methyl-group acceptance activities (Figure 8). The results with the tRNA Asn , tRNA Thr , tRNA Trp , tRNA Ala , tRNA Cys , tRNA Pro and tRNA Val UAC transcripts consisted of their in vivo m 2 G10 modification patterns (Figure 2): the tRNA Asn , tRNA Thr , tRNA Trp and tRNA Val UAC transcripts were methylated by Trm11-Trm112 but tRNA Ala , tRNA Cys and tRNA Pro were not methylated. However, unexpectedly, we found that tRNA Val AAC1 was methylated very slowly. In Figure 8, the slow methylation of tRNA Val AAC1 is visualized using the gel assay (transcript 46). Therefore, we decided that tRNA Val AAC1 should be analyzed further, and these experiments are described later in this paper.

U38 in tRNA Ala Is a Negative Element for Methylation by Trm11-Trm112
The presence of an intron disturbs the methylation by Trm11-Trm112 (transcript 2 in Figure 3). This result suggests that Trm11-Trm112 recognizes the anticodon-loop. For tRNA Ala and tRNA Cys , we considered whether the sequence of anticodon-loop might act negatively in methylation by Trm11-Trm112.
We prepared nine mutant tRNA transcripts to investigate the anticodon-loop of tRNA Ala (transcripts 51-59 in Figure 9). The sequences of mutant tRNA transcripts are shown in Figure 9A. Transcripts 51-57 are chimera tRNA transcripts of tRNA Phe (transcript 1, black) and tRNA Ala (transcript47, red). As shown in Figure 8, the wildtype tRNA Ala transcript is not methylated by Trm11-Trm112. When the aminoacyl-stem (transcript 51), D-arm (transcript 52) or T-arm (transcript 54) of tRNA Phe was replaced by the corresponding region of tRNA Ala , methylation by Trm11-Trm112 could be clearly observed ( Figure 9B). In contrast, when the anticodon-arm of tRNA Phe was replaced with that of tRNA Ala , the methyl group acceptance activity was completely lost (transcript 53 in Figure 9B). Thus, this result shows that the anticodon-arm of tRNA Ala contains a negative element(s). To clarify whether the anticodon-loop contains the negative element(s), we prepared two chimera tRNA transcripts. When the anticodon-stem of tRNA Phe was replaced with that of tRNA Ala , methylation by Trm11-Trm112 was still observed (transcript 55 in Figure 9C). In contrast, the substitution of the anticodon-loop of tRNA Phe with that of tRNA Ala caused complete loss of methyl group acceptance activity (transcript 56 in Figure 9C). Furthermore, when the anticodon-loop of tRNA Ala was substituted with that of tRNA Phe (transcript 57), this transcript was clearly methylated ( Figure 9D). Thus, these results demonstrate that the negative element(s) for methylation by Trm11-Trm112 in tRNA Ala is contained in the anticodon-loop. In general, the base at position 38 in tRNA is conserved as purine. However, in the case of tRNA Ala , position 38 is U. We prepared two point-mutation tRNA Phe transcripts (transcripts 58 and 59). When G37 in tRNA Phe was substituted by A, this transcript was well methylated (transcript 58 in Figure 9E). In contrast, when A38 in tRNA Phe was substituted by U, this transcript was not methylated (transcript 59 in Figure 9E). Taking these results together, we conclude that U38 in tRNA Ala ( Figure 9F) is a negative element for methylation by Trm11-Trm112.

The U32-A38 Base Pair in tRNA Cys Is a Negative Element for Methylation by Trm11-Trm112
Trm11-Trm112 recognizes the anticodon-loop in tRNA. The anticodon-loop of tRNA Cys is not standard because U32-A38 and U33-A37 base pairs can be formed ( Figure 10A). We considered that these unusual base pairs may act negatively for methylation by Trm11-Trm112. To test this idea, we prepared a point mutation tRNA Cys transcript, in which U32 was substituted by C (transcript 60). Consistent with our hypothesis, as shown in Figure 10B, transcript 60 was methylated by Trm11-Trm112. In contrast, the wild-type tRNA Cys transcript (transcript 48) is not methylated. These results show that the U32-A38 base pair is a negative element for methylation by Trm11-Trm112. It should be mentioned that U32 and A37 are modified to Ψ32 and i 6 A37, respectively, in native tRNA. Therefore, the Ψ32-A38 base pair is probably formed in native tRNA Cys . The precise effect of the Ψ32-A38 base pair on methylation by Trm11-Trm112 is not clear.

A Portion of tRNA Val
AAC1 Is Methylated by Trm11-Trm112 in S. cerevisiae Cells Our biochemical studies on Trm11-Trm112, as described in this report, explain almost all of the m 2 G10 modification patterns observed in S. cerevisiae class I tRNAs: class I tRNAs possess regular-size (5 nt) or shorter variable regions. However, one exception remained: tRNA Val AAC1 . Although tRNA Val AAC1 is reported to possess unmodified G10 [50], our experiments show that this tRNA transcript (transcript 46) is slowly methylated by Trm11-Trm112 (Figure 8). We hypothesized that this tRNA may be methylated by Trm11-Trm112 in S. cerevisiae cells. To confirm this idea, we purified native tRNA Val UAC ( Figure 11A) and tRNA Val AAC1 ( Figure 11B) using the solid-phase DNA probe method [51,52]. We prepared a small RNA fraction using Q-Sepharose column chromatography from which native tRNA Val UAC and tRNA Val AAC1 were purified ( Figure 11C). Initially, we performed primer extension experiments to verify whether G10 is modified. The primer was designed to be complimentary from position36 to position 14 in tRNA. It should be mentioned that m 2 G10 is detectable using the primer extension experiment, although m 2 G can form a Watson-Crick base pair with C. To show this, we used native tRNA Val UAC as a positive control: this tRNA possesses m 2 G10 ( Figure 11A). As shown in Figure 11D, m 2 G10 in tRNA Val UAC paused the reverse transcriptase reaction. Next, we analyzed tRNA Val AAC1 in the same experimental condition. In addition to the paused band derived from m 1 G9, a faint band was observed at position 10 ( Figure 11E). This result suggests that a portion of G10 in tRNA Val AAC1 is modified. In S. cerevisiae tRNAs, m 2 G is only one modification at position 10 ( Figure 2). Therefore, we considered that a portion of tRNA Val AAC1 is methylated by Trm11-Trm112 in vivo. To confirm this idea, we purified tRNA Val AAC1 from wild-type and trm11 gene deletion strains of S. cerevisiae. The modified nucleosides in these tRNAs were analyzed ( Figure 12). In tRNA Val AAC1 from the wild-type strain, a small but clear m 2 G peak was observed ( Figure 12A). In contrast, tRNA Val AAC1 from the trm11 gene deletion strain does not contain m 2 G ( Figure 12B). Thus, the m 2 G modification in tRNA Val AAC1 from the wild-type strain is derived from the activity of Trm11-Trm112. Taking these experimental results together, we conclude that a portion of tRNA Val ACC1 is modified by Trm11-Trm112 in vivo. The modification pattern of tRNA Val AAC1 in S. crevisiae cells coincides with the in vitro experimental result (Figure 8). The ID number of tRNA Val AAC1 from S. crevisiae in the T-psi-C database [43] is tdbR00000464. Our experimental results in this study provide novel additional information about this tRNA. When the peak area of m 1 G of tRNA Val AAC1 from the wild-type strain was expressed as 100%, the peak area of m 2 G was calculated as 13% ( Figure 12A). Therefore, more than 85% of tRNA Val AAC1 possesses unmodified G10, as reported previously [50]. UAC was analyzed using the primer extension method. Because dideoxy DNA sequencing was preformed using the template DNA for transcript 50, the T7 RNA polymerase promoter region can be read in addition to the tRNA coding region. Transcript 50 was methylated by Trm11-Trm112 with non-radioisotope AdoMet and then used for the primer extension. The paused band derived from m 2 G10 is marked. (E) Purified tRNA Val AAC1 was analyzed using the primer extension method. In addition to the paused band derived from m 1 G9, a faint posed band is observed at position 10.

Discussion
For a long time, it has been an enigma why yeast Trm11-Trm112 acts only on a subset of tRNAs. In this study, our biochemical experiments explain the m 2 G10 modification pattern of all class I tRNAs from S. cerevisiae. However, two class II tRNAs (tRNA Leu and tRNA Ser ) were not analyzed. tRNA Leu has the m 2 G10 modification but tRNA Ser does not (Figure 2). Transfer RNA Ser species possess a unique Um44 modification, which is formed by Trm44 [53]. Therefore, there is a possibility that the Um44 modification has a negative effect on m 2 G10 formation by Trm11-Trm112. In this study, we used tRNA transcripts that do not possess modifications. Therefore, we did not investigate class II tRNAs. With tRNA modifications, one modification often affects the formation of other modifications either negatively or positively [2,54]. For example, in the case of S. cerevisiae, the D20 modification mediated by Dus2 [55,56] was not formed in the absence of the Gm18 modification [57]. Furthermore, recently, the order of the modifications (m 5 U54, Ψ55 and m 1 A58) of the T-arm of tRNA Phe has been reported [58]: in yeast cytoplasmic tRNAs, m 5 U54, Ψ55 and m 1 A58 are formed by Trm2 [59,60], Pus4 [61] and the Trm6-Trm61 complex [62], respectively. Moreover, with the anticodon-loop modifications, the modified nucleosides and tRNA modification enzymes form circuits (networks) [63][64][65][66]. For example, the Gm34 modification in tRNA Phe mediated by the Trm7-Trm734 complex [63,[66][67][68] requires Cm32 modification by the Trm7-Trm732 complex [68] and the m 1 G37 modification by Trm5 [69][70][71]. In this view point, the Ψ13 modification, which is synthesized by Pus7 [72,73], may have an effect on the methylation by Trm11-Trm112 because various tRNAs containing Ψ13 do not possess the m 2 G10 modification. In addition, the anticodonloop region contains various hyper modifications (Figure 2 and Reference [1]). Because Trm11-Trm112 recognizes the nucleotide around position 38, these modifications in the anticodon-loop may affect methylation by Trm11-Trm112. To clarify these issues, further study will be necessary.
During the course of this study, Bourgeois et al. proposed a model of interaction between Trm11-Trm112 and tRNA [9]. In their model, Trm112 interacts with the anticodonloop region in a substrate tRNA according to the movement of the THUMP domain in Trm11. Our biochemical experimental results described in the current study support this model because Trm11-Trm112 recognizes the nucleotide around position 38. To clarify the molecular details, a crystal structure of a Trm11-Trm112 and tRNA complex is required.
The physiological role of the m 2 G10 modification of tRNA in eukaryotic cells is not clear although m 2 G10 probably stabilizes the D-stem structure. In fact, human disease derived from a defect of m 2 G10 modification has not been reported [74]. A double deletion strain of the S. cerevisiae trm1 and trm11 genes showed growth retardation [4]. Therefore, the m 2 G10 modification probably works coordinately with other modifications such as m 2 2 G26. A similar phenomenon has been reported: a double mutant strain of the S. cerevisiae trm4 and trm8 genes showed temperature-sensitive growth [75]. Trm4 and Trm8 is a multisite-specific tRNA m 5 C methyltransferase [76] and the catalytic subunit of tRNA m 7 G46 methyltransferase [77], respectively. Thus, in S. cerevisiae cells, the modifications in the three-dimensional core of tRNA probably works coordinately. Trm112 is a hub protein that activates multiple methyltransferases [6,26]. Therefore, the formation of the Trm11 and Trm112 complex has effects on the activities of other methyltransferases in S. cerevisiae cells. If the amount of Trm11 is increased, the activities of other methyltransferases are downregulated, and this effect is not negligible. To understand the physiological role of the m 2 G10 modification and Trm11-Trm112, further study is necessary.

Conclusions
In this study, we clarified the elements in class I tRNAs from S. cerevisiae required for methylation by Trm11-Trm112. The results are summarized in Figure 13. Precursor tRNA does not act as a substrate for Trm11-Trm112. The presence of a 5 -leader sequence, intron and 3 -trailer sequence negatively affect methylation by Trm11-Trm112. The CCA terminal region is essential for the methylation by Trm11-Trm112. In the CCA terminus, A76 (magenta in Figure 13) is essential but C74 and C75 (orange in Figure 13) are not required for methylation by Trm11-Trm112. Furthermore, a deletion of the CCA terminus causes loss of methylation by Trm11-Trm112. Therefore, the phosphate ribose backbone of C74 and C75 region is required for methylation by Trm11-Trm112. Trm11-Trm112 methylates standard tRNAs. Transfer RNAs that possess a regular-size variable region (5 nt: blue in Figure 13) are methylated by Trm11-Trm112. Therefore, six tRNA species (tRNA Asp , tRNA Glu , two tRNA Gly , and two tRNA His ), which possess short variable regions, are not substrates for Trm11-Trm112. Trm11-Trm112 requires the G10-C25 base pair for methylation. In Figure 13, G10 and C25 are colored in red and magenta, respectively. Therefore, five tRNAs (tRNA Glu , two tRNA Gly and two tRNA Pro ) that possess the G10-U25 base pair are not methylated by Trm11-Trm112. Furthermore, Trm11-Trm112 recognizes the anticodon-loop region. Transfer RNA Ala and tRNA Cys possess a non-standard anticodon-loop. In the case of tRNA Ala , U38 acts negatively in methylation by Trm11-Trm112. In the case of tRNA Cys , the unusual U32-A38 base pair disturbs the methylation by Trm11-Trm112. In S. cerevisiae, tRNA introns are inserted between positions 38 and 39. Therefore, these experimental results suggest that Trm11-Trm112 interacts with the nucleotide around position 38. The interaction between the T-arm and D-arm is probably required for the maintenance of the correct distances and angles between the CCA terminus, anticodon-loop and methylation site (G10). As shown in Figure 13, Trm11-Trm112 probably interacts with substrate tRNA from the inside of the L-shaped tRNA structure. In addition, our in vitro experiment showed that the tRNA Val AAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNA Val AAC1 from the wild-type and trm11 gene deletion strains and confirmed that a portion (10-13%) of tRNA Val AAC1 is methylated by Trm11-Trm112 in S. cerevisiae cells. Thus, our biochemical studies described in this report explain the m 2 G10 modification pattern of all class I tRNAs from S. cerevisiae and elucidates the Trm11-Trm112 binding sites in tRNA.

Construction of Trm11-Trm112 Expression System in E. coli Cells
Cloning of trm11 and trm112 genes has been previously reported [45]. Briefly, the trm112 gene was fused to the His x 6 tag and HRV3C protease cleavage site in pET21a (Novagen, Birmingham, UK). The trm11 gene with T7 RNA polymerase promoter was inserted down-stream from the trm112 gene. The constructed plasmid was named pET21a-6x His-Trm112-Trm11.

Expression of Trm11-Trm112 in E. coli Cells
The pET21a-6x His-Trm112-Trm11 plasmid was introduced into E. coli BL21(DE3) Rosetta 2 strain. The transformant was cultured at 37 • C in 1 L of LB medium containing 100 µg/mL ampicillin until the optical density at 600 nm reached 0.6. The culture was cooled on ice for 1 h, and then, isoproypl-β-D-thiogalactopyranoside and ZnCl 2 were added (final concentrations of 1 mM and 100 µM, respectively). The culture was further incubated at 20 • C for 48 h. The cells were collected by centrifugation at 4320× g at 4 • C for 20 min, frozen in liquid nitrogen and stored at −80 • C before use.

Preparation of tRNA Transcripts
Transfer RNA transcripts were prepared using an in vitro T7 RNA polymerase reaction, as described previously [78]. We used 2 -O-methylated DNA primers for construction of template DNAs. For example, the template DNA for tRNA Phe transcript was constructed using the following primers: tRNA Phe forward primer, 5 -GGG TAA TAC GAC TCA CTA TAG CGG ATT TAG CTC AGT TGG GAG CGC CAG ACT GAA GAT CTG GAG GTC -3 ; tRNA Phe reverse primer, 5 -TmGG TGC GAA TTC TGT GGA TCG AAC ACA GGA CCT CCA GAT CTT CAG TCT GG-3 . The transcripts were purified using Q-Sepharose column chromatography and 10% PAGE (7 M urea).

Measurement of Activity of Trm11-Trm112
Typical assay conditions were as follows: 0.10 µM Trm11-Trm112, 10.0 µM tRNA transcript and 20.1 µM AdoMet (20 mM non-radioisotope labeled AdoMet and 0.06 mM 3 H-AdoMet) in 30 µL of buffer C were incubated at 30 • C for 2 min, and then, 20 µL of sample was used for the filter assay. To visualize the methylation, 14 C-AdoMet was used. Then, 0.10 µM Trm11-Trm112, 10.0 µM tRNA transcript and 20.0 µM 14 C-AdoMet in 30 µL of buffer C were incubated at 30 • C for 2 min, and then, RNA was recovered by phenol-chloroform treatment and ethanol precipitation. The RNA was dissolved in 20 µL of water, and then, 0.025 A260 units of RNA was loaded onto a 10% polyacrylamide gel containing 7 M urea. After the electrophoresis, the gel was stained with methylene blue and dried. The incorporation of the 14 C-methyl groups was monitored with a FLA-2000 (GE Healthcare, Tokyo, Japan) imaging analyzer.

Sacharomyces cerevisiae Strains and Culture
A block of wild-type S. cerevisiae (a block of baker's yeast, 200 g) was purchased from a local bakery in Japan. The Saccharomyces cerevisiae trm11 gene deletion strain was purchased from Funakoshi (Tokyo, Japan, code: YSC6273-201937501). The trm11 gene deletion strain was cultured in 1 L of YPD medium at 30 • C for 10 h, and then, the cells were collected by centrifugation at 4320× g at 4 • C for 30 min. The cells were frozen by liquid nitrogen and stored at −80 • C before use.

Preparation of Small RNA Fraction from S. cerevisiae Cells
Wet cells (50 g) were suspended in 250 mL TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA), and then, 250 mL TE buffer-saturated phenol was added. The suspension was shaken at 37 • C for 2 h and then centrifuged at 6000× g at 4 • C for 10 min. The aqueousphase was collected, and then, an equal volume (200 mL) of TE buffer-saturated phenol was added. The sample was shaken at 37 • C for 30 min and then centrifuged at 6000× g at 4 • C for 10 min. Total RNA was prepared from the aqueous-phase (200 mL) by ethanol precipitation. The total RNA was dissolved in 40 mL of buffer D (20 mM Tris-HCl (pH 7.6), 400 mM NaCl) and loaded onto a 10 mL Q-Sepharose column. RNAs were separated by a 400-1000 mM NaCl linear-gradient in buffer D. The small RNA fractions were assessed by 10% PAGE (7 M urea), combined and recovered by ethanol precipitation.

Purification of tRNA Val
UAC and tRNA Val AAC1 Using the Solid-Phase DNA Probe Method Native tRNA Val UAC and tRNA Val AAC1 were purified from the small RNA fraction using the solid-phase DNA probe method, as reported previously [52]. Hybridization of tRNA and DNA probe was performed by cooling from 69 • C to 65 • C over 10 min. The sequences of DNA probes are as follows: for tRNA Val UAC , 5 -GTA AAG GCG ATG TCT TGA ACC AC-biotin 3 ; for tRNA Val AAC1 , 5 -GTTAAG CAG ATG CCA TAA CCG AC-biotin 3 . The probes were designed to be complementary from position 36 to position 13 in the tRNA. Because tRNA Val UAC possesses m 2 2 G26, we used T instead of C as the corresponding complementary sequence.

Primer Extension
Primer sequences for primer extension are as follows: for tRNA Val UAC , 5 -GTA AAG GCG ATG TCT TGA ACC ACT-3 ; for tRNA Val AAC1 , 5 -GTC AAG CAG ATG CCA TAA CCG ACT-3 . The primers were purified by 10% PAGE (7 M urea) before 5 -32 P-labeling to remove short byproducts. Then, 50 pmol of the primer was incubated with 20 units of T4 polynucleotide kinase (Takara, Kyoto, Japan) and 1 µL of γ-32 P-ATP at 37 • C for 1 h. The 5 -32 P-labeled primer was purified by 10% PAGE (7 M urea) again. Dideoxy DNA sequencing was performed using a Promega dideoxy DNA sequencing kit (Promega, Madison, WI, USA). The template DNAs for T7 RNA polymerase transcription were used as the template DNA for dideoxy DNA sequencing. Then, 0.02 A260 units of purified tRNA Val UAC (or tRNA Val AAC1 ), 1.0 pmol 5 -32 P-labeled primer, 1 mM EDTA (pH 8.0) and 1 mM of each dNTP mixture (total volume was 30 µL) were incubated at 90 • C for 5 min to denature the tRNA structure and then cooled on ice. A total of 4 µL of 5 × Prime Script buffer (Takara, Kyoto, Japan), 200 units of Prime Script reverse transcriptase (Takara, Kyoto, Japan, code: 2680A) and 1 µL of RNasin (Promega, RNase inhibitor, code: N2111) were added to the sample. The sample was incubated at 45 • C for 60 min and then heated at 70 • C for 15 min. The reverse transcriptase reaction was stopped by adding 20 µL of the gel loading solution (0.02% xylene cyanole, 0.02% bromophenol blue and 7 M urea). The sample was separated by 15% PAGE (7 M urea). The gel was dried, and the autoradiogram of the gel was obtained using a FLA-2000 (GE Healthcare) imaging analyzer.

Nucleoside Analysis of Purified tRNA
Nucleoside analysis of purified tRNA was performed as described previously [79]. Purified tRNA Val UAC (0.60 A260 units) or tRNA Val AAC1 (0.45 A260 units) were digested with 2 µg RNase A, 4 µg snake venom phosphodiesterase and 0.5 units of bacterial alkaline phosphatase in 20 µL of 50 mM Tris-HCl (pH 8.0) at 37 • C for 24 h, and then, nucleosides were analyzed using a Hitachi L-2000 HPLC system equipped with a reverse-phase C18 column (Nucleosil 100 C18: 25 cm × 4.6 mm, 7 µm; GL Science, Inc., Tokyo, Japan). The elution positions of modified nucleosides (m 1 A, m 7 G, m 5 C, I and m 5 U) were confirmed using standard markers (Sigma). The elution times of m 1 G and m 2 G were determined based on enzymatic formation using Aquifex aeolicus TrmD [80] and Trm1 [81], respectively.